Lin+ Identification of CD68

Identification of CD68+lin− Peripheral Blood Cells with Dendritic Precursor Characteristics Herbert Strobl, Clemens Scheinecker, Elisabeth Riedl, Bettina Csmarits, Concha Bello-Fernandez, Winfried F. Pickl, Otto This information is current as Majdic and Walter Knapp of September 28, 2021. J Immunol 1998; 161:740-748; ; http://www.jimmunol.org/content/161/2/740 Downloaded from References This article cites 58 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/161/2/740.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Identiﬁcation of CD68؉lin؊ Peripheral Blood Cells with Dendritic Precursor Characteristics1

Herbert Strobl,2* Clemens Scheinecker,*‡ Elisabeth Riedl,* Bettina Csmarits,† Concha Bello-Fernandez,* Winfried F. Pickl,† Otto Majdic,† and Walter Knapp*†

Expression of CD68 (macrosialin) in the absence of surface and lysosomal lineage marker molecules is a characteristic feature of T zone-associated plasmacytoid monocytes, which were recently shown to represent precursors of dendritic cells (DC). We dem- ,onstrate here a minor population of strongly CD68-positive (CD68bright ) blood cells that lack all analyzed myeloid surface (CD14؊ CD33؊, CD13؊, CD11b؊, CD11c؊) and lysosomal (myeloperoxidase, MPO؊ and lysozyme, LZ؊) marker molecules (0.4 ؎ 2% of the total mononuclear cells). These CD68bright, lineage marker-negative (lin؊) cells can be induced to proliferate in the presence of IL-3. They do not acquire myeloid features even upon stimulation with granulocyte-macrophage CSF plus IL-1, IL-3, and IL-6. Downloaded from Instead, these cells develop typical DC characteristics upon culture. Furthermore, these CD68brightlin؊ DC precursors acquire mature DC characteristics (CD86؉, CD83؉, CD54bright ) upon stimulation with CD40 ligand plus IL-3. A second subset of DC precursor-like blood cells was found to weakly express CD68 (0.3 ؎ 0.2% of the total mononuclear cells) and to coexpress several myeloid lineage associated molecules (LZ؉, CD11c؉, CD33؉, CD13؉). Cells of this second subset resemble both previously described myeloid-related peripheral blood DC and germinal center DC. Analysis of peripheral blood leukocytes for CD68 thus revealed the existence of two cell subsets that phenotypically resemble lymphoid tissue-associated DC. The unique phenotype http://www.jimmunol.org/ -CD68brightlin؊ is highly reminiscent of T zone-associated plasmacytoid monocytes. CD68brightlin؊ blood leukocytes also function ally resemble plasmacytoid monocytes. The lack of all analyzed myeloid features by CD68brightlin؊ blood leukocytes suggests that these cells arise from a novel nonmyeloid human DC differentiation pathway. The Journal of Immunology, 1998, 161: 740–748.

he recently cloned lysosomal/endosomal molecule CD68/ (19). This absence of lineage-associated marker molecules to- macrosialin (1–3) represents a type I integral membrane gether with the expression of the mucin-like lysosomal mem- T protein with significant sequence homology of the membrane protein CD68 reminded us of DC found in lymph nodes brane proximal and cytoplasmic domains to a family of lysosomal/ and skin (12–14). plasma membrane shuttling proteins known as the lamp/lgp family Minute numbers of cells with DC precursor characteristics have by guest on September 28, 2021 (4–8). CD68/macrosialin is heavily O-glycosylated and is classi- been demonstrated previously in MNC fractions of peripheral fied as a mucin-like membrane protein. Oxidized low density li- blood (20–29). They were described as HLA-DRϩCD4ϩlinϪ leu- poprotein has recently been identified as a putative ligand of kocytes that comprise at least two phenotypically and functionally CD68 (9, 10). differing subsets. One subset coexpresses the granulomonocyte- CD68 represents a classical and widely used immunohistologic associated molecules CD33, CD13, and CD11c. Phenotypically marker molecule for cells of the monocyte/macrophage and den- very similar cells have very recently been demonstrated in germi- 3 dritic cell (DC) system (11–18). Flow cytometric analysis of nal centers of lymphoid tissues (30). In contrast, the other subset is CD68 expression was only recently introduced by us and allowed negatively defined by the absence of or very weak expression of us to identify a small population of about 2% of PBMC that ex- CD13, CD33, and CD11c and phenotypically resembles a recently press intracellular CD68, but lack the monocyte marker CD14 and identified DC precursor population in T cell areas (31) of lymphoid are negative for the lineage-associated marker molecules CD3 tissues. Thus, DC populations in lymphoid tissues might be repop- (T cells), CD19 (B cells), and CD16 (NK cells/neutrophils) ulated by the observed peripheral blood DC precursors. HLA- DRϩlinϪ peripheral blood cells also include various other important leukocyte progenitor/precursor populations, such as CD34ϩ *Institute of Immunology, Vienna International Research Cooperation Center, No- circulating hemopoietic progenitor cells or putative CD4ϩ lym- vartis Forschungsinstitut; †Institute of Immunology; and ‡Department of Internal Medicine III, Division of Rheumatology, University of Vienna, Vienna, Austria phoid precursors, which are difficult to identify using current Received for publication August 1, 1997. Accepted for publication March 23, 1998. procedures (32). Given the enormous functional importance of DC in the induc- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance tion and regulation of immune responses and our limited knowl- with 18 U.S.C. Section 1734 solely to indicate this fact. edge of in vivo DC development, differentiation, and migration, 1 This work was supported by Fonds zur Fo¨rderung der Wissenschaftlichen For- we considered it of substantial interest to analyze the observed ¨ schung in Osterreich. CD68ϩlinϪ candidate DC precursor population in more detail. 2 Address correspondence and reprint requests to Dr. Herbert Strobl, Institute of Im- CD68ϩ peripheral blood leukocytes indeed include two DC pre- munology, Vienna International Research Cooperation Center, Novartis Forschun- gsinstitut, University of Vienna, Brunnerstrasse 59, A-1235 Vienna, Austria. E-mail cursor subsets, and both resemble DC populations in lymphoid address: [email protected] tissues. CD68brightlinϪ cells phenotypically and functionally re- 3 Abbreviations used in this paper: DC, dendritic cells; MNC, mononuclear cells; LZ, semble plasmacytoid monocytes. The second CD68dim DC precur- lysozyme; PE, phycoerythrin; linϪ, lineage marker negative; mdDC, monocyte-derived dendritic cells; GM-CSF, granulocyte-macrophage colony-stimulating factor; sor-like population resembles myeloid-related cells and germinal rh, recombinant human; CD40L, CD40 ligand; MPO, myeloperoxidase. center DC.

FIGURE 1. Intracellular CD68 expression in linϪ MNC. Freshly isolated MNC were stained with a mixture of biotinylated Abs speciﬁc for the lineage molecules CD14, CD3, CD19, and CD16 (LIN) followed by streptavidin PerCP. After subsequent ﬁxation and permeabilization, cells were stained for intracellular CD68 expression (FITC). Dot plots show CD68 (x-axis) against lineage molecule expression (LIN, y-axis) or negative control of ungated MNC. Sixty thousand cells were analyzed.

Materials and Methods (Pharmacia, Uppsala, Sweden). Lineage marker-negative (linϪ) MNC were Antibodies obtained by first removing rosette-forming cells with neuraminidase- Downloaded from treated sheep erythrocytes and then by immunomagnetic depletion, as pre- Murine mAbs of the following specificities were used in our study: CD68 viously described (34), of all cells reactive to a mixture of CD14 (clone (clone Ki-M7, IgG1), human myeloperoxidase (clone H-43-5, IgG1), hu- MEM18), CD11b (clone LM-2), CD3 (clone VIT3b), and CD19 (clone ϩ Ϫ man lysozyme (clone LZ-1, IgG1), CD3 (clone UCHT1, IgG1), and CD14 HD-37) Abs. CD4 lin cells were obtained by FACS sorting of cells dou- (clone MEM18, IgG1) obtained from An der Grub (Kaumberg, Austria); ble stained for CD4 (FITC; clone VIT4) and lineage molecules (PE; CD14, CD19 (clone HD37, IgG1) provided by Dr. B. Do¨rken (Berlin, Germany); CD3, CD19, CD16) using a FACS Vantage flow cytometer (Becton Dick- HLA-DR (clone L243, IgG2a), CD34 (clone HPCA2, IgG1) and CD11c inson). For subsorting on the basis of CD45RA expression, purified

ϩ Ϫ http://www.jimmunol.org/ (clone Leu 12, IgG1) obtained from Becton Dickinson (San Jose, CA); CD4 lin cells were stained for CD45RA (PE) and FACS sorted into ϩ Ϫ ϩ Ϫ Ϫ CD45RA (clone MEM93, IgG1) provided by Dr. W. Horejsi (Prague, CD4 lin CD45RAbright and CD4 lin CD45RA /dim cells. The purity of Czech Republic); EMBP (clone AHE-2, IgG1) provided by Dr. K.M. Sku- all cell populations obtained by sorting was determined by reanalysis by bitz (Minneapolis, MN); CD33 (clone WM-54, IgG1) obtained from Dako FACS and was Ͼ95%. Monocyte-derived DC (mdDC) were generated in (Glostrup, Denmark); Ki-67 (clone MIB-1, IgG1) obtained from Dianova the presence of GM-CSF plus IL-4 with or without TNF-␣ as described (Hamburg, Germany); CD16 (clone 3G8, IgG1) obtained from Caltag (San previously (35, 36). Francisco, CA); and CD13 (clone My7, IgG1) obtained from Coulter (Hi- aleah, FL). Abs specific for CD3 (clone VIT3b, IgG1), CD4 (clone VIT4, Cultivation of MNC subsets IgG2a), CD5 (clone CD5-5D7, IgG1), CD11b (clone LM-2, IgG1), and Purified subsets of linϪ MNC were cultured as described previously (37) CD7 (clone CD7-6B7, IgG2a) were produced in our laboratory. CD54 for up to 7 days at 37°C in a humidified atmosphere and in the presence of

(clone HA58) and CD86 (clone IT2.2.) were obtained from PharMingen by guest on September 28, 2021 5% CO2 in RPMI 1640 medium supplemented with L-glutamine (2.5 mM), (San Diego, CA). CD83 (clone HB15a) was obtained from Immunotech penicillin (125 IU/ml), streptomycin (125 ␮g/ml), and human plasma (Marseille, France). (10%) and in the presence or the absence of the following recombinant human (rh) cytokines: rhGM-CSF (100 ng/ml; Novartis, Basel, Switzer- Immunofluorescence staining land), rhIL-1 (100 U/ml; Novartis), rhIL-6 (10 ng/ml; Novartis) and rhIL-3 Membrane staining. For membrane staining, 50 ␮l of isolated MNC (107/ (100 U/ml; Behring, Marburg, Germany), trimeric human CD40 ligand ml) were incubated for 15 min at 0 to 4°C with 20 ␮l of conjugated mAb. (CD40L) fusion protein (200 ng/ml; provided by Dr. S. D. Lyman, Immu- Triple stainings were performed by first incubating cells with a mixture of nex, Seattle, WA). biotinylated Abs specific for the lineage molecules CD14, CD3, CD19, and Morphologic analysis CD16 together with phycoerythrin (PE)-labeled Abs, then washed twice and subsequently incubated with the second step reagent streptavidin Both freshly isolated and cultured cells were morphologically analyzed in PerCP (Becton Dickinson). Afterward, cells were submitted to intracellular culture vessels using phase contrast microscopy or were cytocentrifuged on staining. microscope slides (2 ϫ 104 cells/slide) using a Cytospin-2 centrifuge Intracellular staining. For suspension stainings of intracellular Ags we (Shandon, Pittsburgh, PA), stained with May-Grunwald-Giemsa, and then used the commercially available reagent combination Fix&Perm from An analyzed by light microscopy. der Grub and followed the proposed procedure. In short, cells were first fixed for 15 min at room temperature (50 ␮l of cells plus 100 ␮l of form- Thymidine incorporation assay aldehyde-based fixation medium). After one washing with PBS, pH 7.2, Ϫ Thymidine incorporation by cultures of purified subsets of lin MNC (5 ϫ cells were resuspended in 50 ␮l of PBS and mixed with 100 ␮l of perme- 103 cells/well) was measured after a total culture period of 72 h in the abilization medium plus 20 ␮l of fluorochrome-labeled Ab. After a further presence or the absence of the above-mentioned cytokines. [3H]thymidine incubation for 15 min at room temperature, cells were washed again and (Amersham, Aylesbury, U.K.) was added to cultures 16 h before harvest- analyzed. ing. Incorporated radioactivity was measured using a Top-Count mi- Indirect immunofluorescence stainings for the proliferation-associated croscintillation counter (Packard, Meriden, CT). nuclear Ag Ki-67 were performed on cytocentrifuged cells using a Cyto- spin-2 centrifuge (Shandon Southern Products, Astmoor, U.K.). Fixation Mixed leukocyte reaction and permeabilization were performed as described previously (33). Graded numbers of irradiated (30 Gy; 137Cs source) stimulator cells (sub- Flow cytometry sets of linϪ MNC) were added to constant numbers (5 ϫ 104/well) of purified (Ͼ98%) allogeneic T cells in round-bottom 96-well tissue culture Flow cytometric analyses were performed with a FACScan flow cytometer plates (Costar, Cambridge, MA). Stimulation of responding T cells was (Becton Dickinson) equipped with a single laser emitting at 488 nm. For monitored by measuring [3H]thymidine incorporation on day 5 of culture analysis of CD68 expression in lineage Ag-negative MNC, data for at least as described above. 60,000 cells were acquired and stored in list-mode files. FACS sortings were performed with a FACS Vantage flow cytometer (Becton Dickinson). Results ϩ Ϫ Cells Identification of CD68 lin MNC Peripheral blood samples were obtained from healthy volunteers and im- Double staining of PBMC for intracellular CD68 (x-axis) vs lin- mediately processed. MNC were isolated by flotation on Ficoll/Hypaque eage marker molecules (mixture of CD14, CD3, CD19, and CD16 742 CD68ϩlinϪ PERIPHERAL BLOOD DENDRITIC PRECURSOR SUBSETS

FIGURE 2. Phenotypic analysis of CD68ϩlinϪ normal adult PBMC. Freshly isolated MNC were combined stained for lineage molecules (CD14, CD3, CD19, and CD16; PerCP), intracellular CD68 (FITC), and several informative molecules (PE). LinϪ cells were Downloaded from gated according to the marker settings shown in Figure 1. Gated linϪ cells are shown. Diagrams represent analyses for CD68 (FITC; x-axes) vs various marker molecules (PE; y-axes). Fluores- cence distributions are representative of ﬁve experiments. Markers were set according to isotype-matched negative control stainings. At http://www.jimmunol.org/ least 60,000 cells were analyzed. by guest on September 28, 2021

Ab conjugates; y-axis) clearly resolved a small population of erably weaker HLA-DR staining than CD68dimlinϪ cells (Fig. 2). CD68ϩlinϪ cells (Fig. 1). The size of this population varied be- The CD68brightlinϪ and CD68dimlinϪ subsets represent, on the av- tween 1.5 and 2.6% (mean, 1.9 Ϯ 0.5%; n ϭ 5) of all MNC. erage (n ϭ 5), 0.4 Ϯ 0.2 and 0.3 Ϯ 0.2%, respectively, of adult blood MNC (Table I). The third CD68ϩ subset clearly distinguish- Phenotypic analysis of CD68ϩlinϪ cells able in this staining profile differs in two respects from the other ϩ Ϫ To further characterize this small CD68 lin cell population, we two subsets. It lacks HLA-DR and has a CD68 staining intensity performed triple staining experiments. For this purpose, one fluo- that lies between those of the two CD68ϩ/HLA-DRϩ populations. rochrome (PerCP) was used for the combined exclusion of the This subset represents 1.2 Ϯ 0.3% of all MNC. lineage-associated molecules CD3, CD19, CD16, and CD14 (as shown in Fig. 1), allowing the use of FITC and PE to analyze linϪ cells for their expression of intracellular CD68 (FITC) and a panel of informative marker molecules (PE). Figure 2 shows a representative phenotypic analysis of such gated CD68ϩlinϪ cells. In total, Table I. Analysis of the distribution of CD68brightlinϪ and CD68dimlinϪ five individual samples were tested. subsets in comparison with CD34ϩ hemopoietic progenitors among total MNC MHC class II molecules (HLA-DR) ϩ Ϫ As shown in Figure 2, CD68 lin cells contain three distinct pop- lin_ Cellsa % MNCb ulations in terms of HLA-DR and CD68 expression. Two popula- CD68ϩ 1.9 Ϯ 0.5 tions, clearly distinct in their CD68 expression density, coexpress CD68bright 0.4 Ϯ 0.2 bright Ϫ HLA-DR. The strongly CD68-positive (termed the CD68 lin CD68dim 0.3 Ϯ 0.2 subset) and the second subset with clearly lower CD68 expression CD34ϩ 0.1 Ϯ 0.01 dim Ϫ density (termed the CD68 lin subset) also differ in their a Ϫ Mononuclear cells negative for CD14, CD3, CD19, and CD16 expression. HLA-DR expression intensity. CD68brightlin cells show consid- b Values expressed as mean Ϯ SD of five experiments. The Journal of Immunology 743

FIGURE 3. Isolation of CD68brightlinϪ and CD68dimlinϪ subsets. The two subsets were isolated from pre-enriched MNC using a sequential two-step ﬂow-sorting procedure (see Mate- rials and Methods). Representative sort window settings are shown. CD4ϩlinϪ cells were isolated (SORT I). Puriﬁed CD4ϩlinϪ cells were stained for CD45RA expression and resorted (SORT II) into CD45RAbright and CD45RAdim fractions, which were identical with CD68brightlinϪ and CD68dimlinϪ subsets, Downloaded from respectively. Cytospin preparations of freshly isolated CD68brightlinϪ and CD68dimlinϪ subsets stained with May-Grunwald-Giemsa are shown. http://www.jimmunol.org/

CD4 molecule CD5 and CD7 molecules by guest on September 28, 2021 The three above-described subsets are also heterogeneous in terms Within the population of CD68ϩlinϪ cells, only CD68dimlinϪ cells of their CD4 expression pattern. The CD68brightlinϪ subset is coexpress CD5. The pan T/NK cell marker molecule CD7 is absent strongly positive for CD4 (Fig. 2). The CD68dimlinϪ subset also from CD68ϩlinϪ cells. expresses CD4, but the intensity is slightly lower. CD68ϩlinϪ cells with intermediate CD68 staining intensity lack CD4 expression. CD34 molecule The proportions of CD68ϩlinϪ cells coexpressing CD4 and CD34 expression was, in all ﬁve experiments, restricted to CD68Ϫ HLA-DR are virtually identical. cells.

CD45RA molecule Lysosomal protein expression ϩ Ϫ In all five experiments the CD68brightlinϪ subset was found to Virtually all CD68 lin cells lack the highly selective pan-granu- strongly express CD45RA, whereas the CD68dimlinϪ subset was lomonocytic lysosomal marker molecule MPO (38). The intracel- CD45RA negative to only weakly positive. lular marker molecule of basophils and eosinophils, eosinophil major basic protein (39) is expressed, but is restricted to CD68ϩLinϪ CD33 and CD13 molecules cells with intermediate CD68 density. Together these data show that the two identified subsets Similar expression patterns were observed for the two GM-asso- CD68brightlinϪ and CD68dimlinϪ share phenotypic characteristics bright Ϫ ciated marker molecules CD33 and CD13. The CD68 lin with DC. subset consistently lacks CD33 and CD13; the CD68dimlinϪ and Ϫ Ϫ the CD68 intermediate density subsets are positive for both mol- Purification of CD68brightlin and CD68dimlin subsets ecules. The highest CD33 expression density was observed in all To further investigate the nature of the CD68brightlinϪ and dim Ϫ experiments for the CD68 lin subset. CD68dimlinϪ subsets we purified these two populations. Because the detection of intracellular CD68 requires fixation and precludes CD11b and CD11c molecules sorting of viable cells, an alternative strategy had to be followed ␤ bright Ϫ dim Ϫ The 2 integrin molecule CD11b is absent from both, for purification of viable CD68 lin and CD68 lin cells. CD68brightlinϪ and CD68dimlinϪ subsets. Cells with intermediate The best way appeared to be FACS sorting of linϪ MNC into CD68 density coexpress CD11b. A somewhat different staining CD4ϩCD45RAbright and CD4ϩCD45RAdim/Ϫ cell fractions. This pattern was found for CD11c. The CD68brightlinϪ subset lacks strategy is based on our observation (see Fig. 2) that CD11c, whereas the CD68dimlinϪ subset is strongly CD11c pos- CD68brightlinϪ and CD68dimlinϪ cells both coexpress CD4, but itive. Cells with intermediate CD68 expression density are CD11c differ in CD45RA expression. CD68brightlinϪ cells are also weakly positive to negative. CD45RAbright; CD68dimlinϪ cells are CD45RA dim to negative. 744 CD68ϩlinϪ PERIPHERAL BLOOD DENDRITIC PRECURSOR SUBSETS

FIGURE 4. Analyses of cultured CD68bright linϪ cells. Flow-sorted CD68brightlinϪ cells were cultured in the presence of cytokines as described in Materials and Methods. Representa- tive cultures are shown (n ϭ 4). Phase contrast photomicrographs (A, low; B, high magnifica- tion) of cells cultured for 7 days in the presence of IL-1, IL-3, IL-6, and GM-CSF are shown. C, Fluorescence microscopy of Ki-67 stainings of cultured cells on day 4 in the presence of IL-1, IL-3, IL-6, and GM-CSF. D, Analysis of the effect of IL-3 or combinations of cytokines on cell proliferation. CD68brightlinϪ cells were stimu- Downloaded from lated with the indicated cytokines for 72 h. [3H]thymidine incorporation was determined as described in Materials and Methods after a final 16-h pulse labeling. Data from one representative experiment are shown. http://www.jimmunol.org/ by guest on September 28, 2021 Figure 3 shows an example of the sort window settings used in thin DC projections (Fig. 4B). In addition, substantial proportions these experiments. Pre-enriched MNC (see Materials and Meth- of cultured cells stimulated with GM-CSF, IL-1, IL-3, and IL-6 ods) were first sorted for CD4ϩlinϪ cells (SORT I). Sorted expressed the proliferation-associated nucleoprotein Ki-67 (21 and CD4ϩlinϪ cells were then stained for CD45RA and sorted again 26% in two experiments on day 4, respectively; Fig. 4C). These for CD45RAbright and CD45RAdim/Ϫ cells (SORT II). Triple stain- observations prompted us to analyze the effects of individual cy- ings for CD68, CD4, and lin molecules, performed in parallel, tokines. We observed that addition of IL-3 alone induces cell cy- confirmed that all CD4ϩlinϪ cells were CD68ϩ and subdivided cling of CD68brightlinϪ cells. These results were confirmed using into CD68bright and CD68dim subsets (data not shown). [3H]thymidine incorporation experiments (Fig. 4D). Total cell numbers stayed approximately constant over the analyzed 7-day bright Ϫ dim Ϫ Morphology of isolated CD68 lin and CD68 lin cells culture period and were equivalent in cultures supplemented with The two subsets of CD68ϩlinϪ MNC clearly differ in their mor- GM-CSF, IL-1, IL-3, and IL-6 or with IL-3 alone (data not Ϫ phologic appearance (see cytospin preparations stained with May- shown). The second, CD68dimlin subset did not show growth Ϫ Grunwald-Giemsa in Fig. 3). CD68brightlinϪ cells are round, with factor dependency similar to that observed for CD68brightlin cells round or lobulated nuclei and abundant cytoplasm. CD68dimlinϪ and did not proliferate. Most of these cells rapidly lost viability, cells are of similar size, but differ from CD68brightlinϪ cells in that and individual cytokines or cytokine combinations did not enhance Ϫ they have a more ruffled cell shape with irregularly shaped and viability. In comparison, cultures of CD68brightlin cells set up in multilobed nuclei. In addition, cells of the CD68brightlinϪ subset parallel clearly showed higher percentages of viable cells, and vi- were more vacuolated than CD68dimlinϪ cells. ability was dependent on whether the culture medium was supplemented with IL-3 (data not shown). Growth characteristics of CD68brightlinϪ and CD68dimlinϪ cells bright Ϫ To further analyze the stage of differentiation and lineage restric- T cell stimulatory capacity of CD68 lin cells tion of the two subsets, we tested their in vitro proliferation and Given the DC-like features of in vitro cultured CD68brightlinϪ differentiation capacities in the presence of cytokines. We first an- cells, we analyzed their capacity to induce allogeneic T cell pro- alyzed the myelopoietic differentiation potential of isolated liferation. As shown in Figure 5, after culture for 7 days in above- CD68brightlinϪ and CD68dimlinϪ subsets using the cytokine com- described GM-CSF-, IL-1-, IL-3-, and IL-6-supplemented me- bination GM-CSF, IL-1, IL-3, and IL-6, which represents a pow- dium, CD68brightlinϪ cells significantly induce allogeneic T cell erful stimulus for growth and myeloid differentiation of hemopoi- proliferation. They are similar in relative potency in the MLR to etic progenitors (37). The following profound changes were DC generated from autologous CD14ϩ monocytes in the presence observed when stimulating purified CD68brightlinϪ cells with this of GM-CSF plus IL-4 stimulation (mdDC), but are less efficient growth combination. Within 48 h large aggregates were formed inducers of T cell proliferation than autologous mdDC generated (Fig. 4A). This was followed on day 5 by the development of long in parallel in the presence of GM-CSF, IL-4, and TNF-␣. The Journal of Immunology 745

Lack of granulomonocytic features of in vitro differentiated CD68brightlinϪ cells Freshly isolated CD68brightlinϪ cells, in marked contrast to CD68dimlinϪ cells, show no specific features of granulomonocytic cells. They express neither MPO (Fig. 2), a hallmark molecule of granulomonocytic differentiation (38), nor LZ (Fig. 6A), a molecule constituitively expressed by granulomonocytic cells that is up-regulated in monocytes and macrophages upon activation (40– 43). Even upon culture of CD68brightlinϪ cells with the granulomonopoietic growth combination GM-CSF plus IL-1, IL-3, and IL-6, we were unable to detect LZ or MPO in these cells (Fig. 6B). Similarly, no induction of expression of the monocyte-associated surface molecules CD14, CD11c, and CD33 occurred (Fig. 6B). The molecular features of in vitro cultured (differentiated) CD68brightlinϪ cells, therefore, are clearly different from those of freshly isolated CD68dimlinϪ cells. The CD68dimlinϪ population expresses LZ and is positive for CD11c and CD33 (Figs. Downloaded from 2 and 6A). FIGURE 5. Immunostimulatory capacity of in vitro cultured bright Ϫ ϫ 4 CD68 lin cells. Purified allogeneic T cells (5 10 /well) were cul- bright Ϫ tured with graded numbers of irradiated CD68brightlinϪ stimulator cells. Expression of costimulatory molecules by CD68 lin and dim Ϫ Stimulator cells are CD68brightlinϪ cells after culture for 7 days in the CD68 lin cells presence of IL-1, IL-3, IL-6, and GM-CSF. DC generated in parallel from bright Ϫ dim Ϫ ϩ Both CD68 lin and CD68 lin cell subsets lack significant http://www.jimmunol.org/ autologous CD14 monocytes (mdDC) in the presence of GM-CSF plus expression of the T cell costimulatory molecule CD86, but they are IL-4 or in the presence of GM-CSF plus IL-4 and TNF-␣ are compared. Negative controls represent unstimulated T cell-depleted autologous MNC clearly CD54 positive. Furthermore, both subsets lack expression (freshly isolated and frozen). of the mature DC marker molecule CD83 (see Fig. 7). by guest on September 28, 2021

FIGURE 6. Analysis of intracellular LZ and other GM-associated molecules. A, LZ expression in fresh CD68brightlinϪ and CD68dimlinϪ subsets. MNC were pre-enriched by sheep erythrocyte rosetting and immunomagnetic depletion as described in Materials and Methods. Lin-depleted fractions were stained for CD68 (FITC), LZ (PE), and the lineage Aga CD3, CD19, CD14, and CD16 (PerCP). Dot plots show gated linϪ cells analyzed for CD68 vs LZ expression. B, Expression of LZ-, MPO-, and GM-associated surface molecules by cultured CD68brightlinϪ cells. CD68brightlinϪ cells were puriﬁed by two-step ﬂow sorting and were cultured for 7 days in the presence of IL-1, IL-3, IL-6, and GM-CSF as described in Materials and Meth- ods. Histograms show the expression of the lysosomal proteins LZ, MPO, and CD68 as well as the surface membrane molecules CD14, CD11c, and CD33. 746 CD68ϩlinϪ PERIPHERAL BLOOD DENDRITIC PRECURSOR SUBSETS

FIGURE 7. Analysis of costimulatory molecules and CD83. MNC were pre-enriched by sheep erythrocyte rosetting and immunomagnetic deple- Downloaded from tion as described in Figure 6A. Lin-depleted fractions were triple stained for CD68 (FITC), CD86, CD83, CD54, or isotype-negative control (PE) and the lineage Ags CD3, CD19, CD14, and CD16 (PerCP). Dot plots show gated linϪ cells analyzed for CD68 vs isotype-negative control, CD86, CD83, or CD54. Data are representative of four experiments. http://www.jimmunol.org/ FIGURE 8. CD40L up-regulates costimulatory molecules and induces CD83 expression on CD68brightlinϪ cells. CD68brightlinϪ cells were puri- CD40 ligation induces expression of CD83 and up-regulation of bright Ϫ fied by sequential immunomagnetic depletion and flow sorting as described costimulatory molecules on CD68 lin cells in Materials and Methods. Freshly isolated cells were stimulated for 7 days Grouard et al. (31) recently demonstrated that isolated tonsil plas- in the presence of IL-3 or IL-3 plus CD40L and then analyzed for the macytoid monocytes can be induced by CD40L plus IL-3 stimu- expression of CD86, CD83, and CD54 molecules as described in Materials lation to acquire features of mature DC. CD68brightlinϪ peripheral and Methods. Dotted lines represent isotype-matched controls; faint lines and bold lines show stainings with specific Abs as indicated. Data are blood leukocytes identified in our study share unique immunophe-

representative of three experiments. by guest on September 28, 2021 notypic (CD68bright linϪ, MPOϪ,LZϪ) and functional features (IL-3-dependent growth) with plasmacytoid monocytes, suggesting that they may represent immediate precursors of plasmacytoid Ϫ The most striking finding of our study is that this CD68brightlin monocytes. Therefore, we analyzed the effect of CD40L costimu- bright Ϫ DC subset clearly differs from granulomonopoietic cells and from lation on the acquisition of mature DC features by CD68 lin dim Ϫ bright Ϫ the other, CD68 lin DC subset based on the lack of expression cells. As shown in Figure 8, stimulation of CD68 lin cells for Ϫ of all analyzed granulomonocyte-associated intracellular (MPO , 7 days with IL-3 plus CD40L significantly induces up-regulation Ϫ Ϫ Ϫ Ϫ LZ ) and cell surface (CD33 , CD13 , CD11c ) molecules. Fur- of CD86 and CD54 molecules, and most cells become positive for Ϫ thermore, CD68brightlin DC are functionally distinguishable from the mature DC marker molecule CD83. In contrast, in the presence granulomonopoietic precursors in that they fail to acquire MPO, of IL-3 alone, the majority of cells remain CD86 negative, and LZ, CD33, CD14, and CD11c expression when stimulated in the virtually all cells remain CD83 negative. presence of cytokines (37). We further observed that cells with this unique myeloid marker-negative (myϪ) CD68brightlinϪ phenotype Discussion can survive and enter cell cycling if they are stimulated in vitro Using intracellular flow cytometric detection of CD68, which rep- with IL-3. Other cytokines or cytokine combinations previously resents a sensitive marker molecule for cells of the granulomono- shown to be important for in vitro DC development from progen- cyte/macrophage and DC system, we identified in this study two itors or monocytes, including GM-CSF, IL-1, IL-4, IL-6, TNF-␣, subsets of DC-like cells in human mononuclear cells. Both subsets and TGF-␤, do not replace IL-3 in this function (data not shown). express CD68 and lack expression of the lineage-associated sur- Thus, on the basis of a bright intracellular CD68 expression pat- face Ags CD14, CD3, CD19, and CD16. CD68ϩlinϪ cells make tern, we describe here a distinct subset of IL-3-responsive DC up, on the average, 1.9 Ϯ 0.5% of the total MNC and include, apart precursors that lack all analyzed lineage features of granulomono- from the two DC-like subsets, basophils, which are known to poietic cells, suggesting that they arise from a nonmyeloid pro- coisolate with MNC in the Ficoll/Hypaque gradient separation progenitor cell differentiation pathway. cedures used here (44, 45). The two DC-like populations identified We show that the absence of expression of the intracellular ly- among CD68ϩlinϪ cells clearly differ in CD68 expression. One sosomal protein LZ clearly distinguishes CD68brightlinϪ cells from subset is weakly CD68ϩ (CD68dimlinϪ cells; 0.3 Ϯ 0.2% of the granulomonocytic cells as well as from the other CD68dimlinϪ total MNC), whereas the other is strongly CD68ϩ (CD68brightlinϪ subset. The distribution of LZ is of particular relevance for our cells; 0.4 Ϯ 0.2% of the total MNC). Among hemopoietic cells, a study, since LZ is known as a highly sensitive and specific intra- strong CD68 expression pattern like that observed for the latter, cellular lineage marker molecule for granulomonopoietic cells (40, CD68brightlinϪ peripheral blood DC subset can only be found in 42). Using a detection method identical with described in this MPOϩ myeloid bone marrow precursors and in CD14ϩ study, we previously observed that LZ protein expression is rap- monocytes (19). idly induced during monopoietic differentiation and even precedes The Journal of Immunology 747 acquisition of (pro)monocyte morphology by hemopoietic progen- gration, is suggested from the higher frequency of these cells under itors (37). The observed distribution of LZ among the two subsets certain pathologic conditions associated with reactive T cell infil- of DC included in the CD68ϩlinϪ population is interesting, since tration or extravasation (50, 57–60). it shows a striking correlation with the distribution of the surface marker molecules CD33, CD13, and CD11c. Only CD68dimlinϪ Acknowledgments peripheral blood DC express these myeloid-related molecules. Fur- We are grateful to A. Renner for sorting cells on the FACS Vantage, ther analysis of these myϩCD68dimlinϪ peripheral blood DC ϩ ϩ bright M. Waclavicek for MLR analyses, and Immunex Corp. (Seattle, WA) for showed additional features (i.e., CD4 , CD5 , HLA-DR , providing CD40 ligand. CD45RA dim to neg, and ruffled cell shapes) previously described characteristic of myeloid peripheral blood DC precursors (23, 25, References 46) and germinal center DC (30). The expression pattern of LZ 1. Holness, C. L., and D. L. Simmons. 1993. Molecular cloning of CD68, a human thus further supports the concept that these DC are myeloid in macrophage marker related to lysosomal glycoproteins. Blood 81:1607. origin (22). 2. Holness, C. L., R. P. da Silva, J. Fawcett, S. Gordon, and D. L. Simmons. 1993. The second, myϪCD68brightlinϪ DC precursor-like subset Macrosialin, a mouse macrophage-restricted glycoprotein, is a member of the lamp/lgp family. J. Biol. Chem. 268:9661. clearly shares characteristics with previously described peripheral 3. Martinez Pomares, L., N. Platt, A. J. Knight, R. P. da Silva, and S. Gordon. 1996. blood leukocyte populations. They are phenotypically similar to Macrophage membrane molecules: markers of tissue differentiation and hetero- previously described “immunologically immature” peripheral geneity. Immunobiology 195:407. Ϫ ϩ Ϫ dim Ϫ 4. Lippincot-Schwartz, J., and D. M. Fambrough. 1987. Cycling of the integral blood DC precursors (lin HLA-DR CD11c , CD33 / , membrane glycoprotein, LEP100, between plasma membrane and lysosomes: Downloaded from CD13dim/Ϫ) (23, 24, 27) and closely resemble recently described kinetic and morphologic analysis. Cell 49:669. Ϫ 5. Fukuda, M., J. Viitala, J. Matteson, and S. R. Carlsson. 1988. Cloning of cDNAs CD2 peripheral blood DC precursors (29). Positive identification encoding human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. based on bright intracellular CD68 expression as shown in our J. Biol. Chem. 263:18920. study clearly distinguishes CD68brightlinϪ blood DC from early 6. Fukuda, M. 1991. Lysosomal membrane glycoproteins. J. Biol. Chem. ϩ 266:21327. lymphoid cells (19) and from CD34 circulating hemopoietic pro- 7. Chen, J. W., Y. Cha, K. U. Yuksel, R. W. Gracy, and J. T. August. 1988. Isolation genitor cells, which are both negative or only weakly CD68 pos- and sequencing of a cDNA clone encoding lysosomal membrane glycoprotein itive. We demonstrate that CD34ϩ cells are, on the average, four- mouse lamp-1. J. Biol. Chem. 263:8754. http://www.jimmunol.org/ 8. Howe, C. L., B. L. Granger, M. Hull, S. A. Green, C. A. Gabel, A. Helenius, and fold less frequent among total MNC (on the average, 0.1% of the I. Mellman. 1988. Derived protein sequence, oligosaccharides, and membrane total MNC) compared with CD68brightlinϪ DC (Table I). insertion of the 120-kDa lysosomal membrane glycoprotein (lgp120): identification of a highly conserved family of lysosomal membrane glycoproteins. Proc. As described above, based on phenotypic and functional criteria, Natl. Acad. Sci. USA 85:7577. bright Ϫ the subset of CD68 lin DC clearly differs from granulomo- 9. Ramprasad, M. P., W. Fischer, J. L. Witztum, G. R. Sambrano, O. Quehenberger, nopoietic cells. One may speculate, therefore, that these DC orig- and D. Steinberg. 1995. The 94- to 97-kDa mouse macrophage membrane protein that recognizes oxidized low density lipoprotein and phosphatidylserine-rich li- inate from a separate nonmyeloid progenitor cell differentiation posomes is identical to macrosialin, the mouse homologue of human CD68. Proc. pathway (47). Evidence for the existence of such a pathway has Natl. Acad. Sci. USA 92:9580. 10. Ramprasad, M. P., V. Terpstra, N. Kondratenko, O. Quehenberger, and

been presented recently (48). Further studies should analyze by guest on September 28, 2021 D. Steinberg. 1996. Cell surface expression of mouse macrosialin and human whether CD68 represents a useful marker for the identification of CD68 and their role as macrophage receptors for oxidized low density lipopro- putative bone marrow progenitors of CD68brightlinϪ blood DC. tein. Proc. Natl. Acad. Sci. USA 93:14833. bright Ϫ 11. Parwaresch, M. R., H. J. Radzun, H. Kriepe, M. L. Hansmann, and J. Barth. 1986. One key finding of our study is that CD68 lin DC respond Monocyte/macrophage reactive monoclonal antibody Ki-M6 recognizes an intra- in vitro to IL-3 stimulation. We show that IL-3 as a single cytokine cytoplasmic antigen. Am. J. Pathol. 125:141. maintains viability and induces cycling of CD68brightlinϪ cells. 12. Franklin, W. A., D. Y. Mason, K. Pulford, B. Falini, E. Bliss, K. C. Gatter, H. Stein, L. C. Clarke, and J. O. McGee. 1986. Immunohistological Analysis of These features independently observed in our study together with human mononuclear phagocytes and dendritic cells by using monoclonal anti- phenotypic (lack of CD11c, CD13, and CD33 expression) and bodies. Lab. Invest. 54:322. morphologic (round cell shape when freshly isolated) characteris- 13. Hall, P. A., C. J. O’Doherty, and D. A. Levison. 1987. Langerhans cell histio- cytosis: an unusual case illustrating the value of immunohistochemistry in diag- tics are highly reminiscent of a recently characterized population nosis. Histopathology 11:1181. of DC precursors in T cell areas of human tonsils (31). Identical 14. Kelly, P. M., E. Bliss, J. A. Morton, J. Burns, and J. O. McGee. 1988. Mono- clonal antibody EBM/11: high cellular specificity for human macrophages. cells were previously identified in electron microscopy studies as J. Clin. Pathol. 41:510. T-associated plasma cells (49). 15. Davey, F. R., J. L. Cordell, W. N. Erber, K. A. Pulford, K. C. Gatter, and Apart from this, independent evidence presented in our study D. Y. Mason. 1988. Monoclonal antibody (Y1/82A) with specificity towards bright Ϫ peripheral blood monocytes and tissue macrophages. J. Clin. Pathol. 753:753. strongly supports our assumption that CD68 lin blood DC 16. Kreipe, H., H. J. Radzun, M. R. Parwaresh, A. Haislip, and M. L. Hansmann. represent circulating precursors of T-associated plasma cells. Im- 1987. Ki-M7 monoclonal antibody specific for myelomonocytic cell lineage and munohistology studies previously demonstrated that T zone-asso- macrophages in human. J. Histochem. Cytochem. 35:117. 17. Micklem, K., J. Cordell, E. Rigney, D. Simmons, K. Pulford, P. Stross, and ciated plasmacytoid cells (plasmacytoid T cells) are discriminable D. Mason. 1989. A macrophage-associated antigen defined by five mAb. In Leu- from other lymphoid tissue-associated macrophage or DC popu- kocyte Typing. IV. White Cell Differentiation Antigens. W. Knapp, B. Do¨rken, ϩ W. R. Gilks, E. P. Rieber, R. E. Schmidt, H. Stein, and A. von dem Borne A., eds. lations in that they are brightly CD68 (50–52) (later renamed Oxford University Press, Oxford, pp. 841–843. plasmacytoid monocytes based on bright CD68 staining (51)), but 18. Weiss, L., D. Arber, and K. Chang. 1994. CD68 A review. Appl. Immunohisto- negative for the classical myelomonocytic lineage marker mole- chem. 2:2. Ϫ bright 19. Strobl, H., C. Scheinecker, B. Csmarits, O. Majdic, and W. Knapp. 1995. Flow cules LZ (52–54) and MPO (52, 55). This unique lin CD68 / cytometric analysis of intracellular CD68 molecule expression in normal and Ϫ Ϫ LZ /MPO phenotype also defines the small population of malignant haematopopiesis. Br. J. Haematol. 90:774. CD68brightlinϪ blood DC in our study. Phenotypic resemblance of 20. Freudenthal, P. S., and R. M. Steinman. 1990. The distinct surface of human bright Ϫ blood dendritic cells, as observed after an improved isolation method. Proc. Natl. the CD68 lin blood DC described here with plasmacytoid Acad. Sci. USA 87:7698. monocytes in lymphoid tissues is further supported by coexpres- 21. Egner, W., R. Andreesen, and D. N. J. Hart. 1993. Allostimulatory cells in fresh sion of HLA-DR, CD4, and CD45RA (50, 51, 54, 55). human blood: heterogeneity in antigen-presenting cell populations. Transplanta- tion 56:945. Typical location of plasmacytoid monocytes around high endo- 22. Thomas, R., L. S. Davis, and P. E. Lipsky. 1993. Isolation and characterization thelial venules (56) further argues that blood precursors continu- of human peripheral blood dendritic cells. J. Immunol. 150:821. 23. Thomas, R., and P. E. Lipsky. 1994. Human peripheral blood dendritic cell sub- ously repopulate these cells. A high turnover rate of plasmacytoid sets: isolation and characterization of precursors and mature antigen-presenting monocytes, probably regulated by the rate of precursor cell immi- cells. J. Immunol. 153:4016. 748 CD68ϩlinϪ PERIPHERAL BLOOD DENDRITIC PRECURSOR SUBSETS

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